Microglia Mediate Contact-Independent Neuronal Network Remodeling via Secreted Neuraminidase-3 Associated with Extracellular Vesicles

Neurons communicate with each other through electrochemical transmission at synapses. Microglia, the resident immune cells of the central nervous system, modulate this communication through a variety of contact-dependent and -independent means. Microglial secretion of active sialidase enzymes upon exposure to inflammatory stimuli is one unexplored mechanism of modulation. Recent work from our lab showed that treatment of neurons with bacterial sialidases disrupts neuronal network connectivity. Here, we find that activated microglia secrete neuraminidase-3 (Neu3) associated with fusogenic extracellular vesicles. Furthermore, we show that Neu3 mediates contact-independent disruption of neuronal network synchronicity through neuronal glycocalyx remodeling. We observe that NEU3 is transcriptionally upregulated upon exposure to inflammatory stimuli and that a genetic knockout of NEU3 abrogates the sialidase activity of inflammatory microglial secretions. Moreover, we demonstrate that Neu3 is associated with a subpopulation of extracellular vesicles, possibly exosomes, that are secreted by microglia upon inflammatory insult. Finally, we demonstrate that Neu3 is necessary and sufficient to both desialylate neurons and decrease neuronal network connectivity. These results implicate Neu3 in remodeling of the glycocalyx leading to aberrant network-level activity of neurons, with implications in neuroinflammatory diseases such as Parkinson’s disease and Alzheimer’s disease.


Materials and Methods.
Cell culture BV2 murine microglia (a kind gift from T. Wyss-Coray) were propagated in DMEM supplemented with 10% hiFBS.Microglia were maintained at a low passage number (< 10 since obtaining initial stocks) and split at or before ~ 70% confluency to avoid runaway inflammation as caused by dead or floating BV2 cells.If cells grew overconfluent, the culture was discarded.Typically, ~ 1e6 microglia were seeded in a T75 in 25 mL split every two days.Microglia were harvested using Gibco enzyme-free dissociation buffer, incubated for 5 min at room temperature, and lifted with gentle tapping and pipetting.Microglia were diluted in an equal volume of complete media, counted with a hemocytometer, pelleted by centrifugation (300 rcf, 5 min), and resuspended in complete media for subculturing.HeLa cells (ATCC CCL-2) were propagated in DMEM supplemented with 10% hiFBS.HeLa cells were subcultured at a confluence below 80% to avoid overgrowth.Typically, HeLa cells were split 1:5 every two to three days.HeLa cells were lifted using trypsin and pelleted by centrifugation (300 rcf, 5 min), before resuspending in fresh media.Preparation of microglia-conditioned media Low passage and subconfluent BV2 microglia were harvested using Gibco enzyme-free dissociation media, incubated for 5 min at room temperature, and lifted with gentle tapping and pipetting.Microglia were diluted in an equal volume of complete media, counted with a hemocytometer, pelleted by centrifugation (300 rcf, 5 min), and resuspended in NB++ media (PN) for subculturing.Cultures for conditioned media were seeded at 4e4 cells per cm 2 at 2e5 cells per mL.In some cases, microglia were stimulated with LPS (1 µg/mL).Cells were cultured at 37 °C in 5% CO 2 for 18 h.The media was then harvested with careful pipetting cleared of floating cells and large debris by centrifugation (500 rcf, 5 min).The clarified media was then transferred to a clean tube.In some cases, 5-N-acetyl-2,3-dehydro-2deoxyneuraminic acid (DANA) was added to 2 mM.An equal volume of conditioned media was then added to neuronal cultures.

Transfection of HeLa cells and conditioning of media
HeLa cells were transfected with plasmids encoding murine Neu3 (wt or the catalytically inactive mutant Y369F) using Transit2020 according to the manufacturer's protocol.Plasmids were custom ordered from Twist Biosciences encoding murine Neu3 (wt or Y369F) C-terminally tagged with a short linker (GSGGGSGGGGSG) followed by a 3xFLAG tag.Constructs were optimized for human codons and cloned into a pCMV vector from Twist.After 24 h, the cells were washed with OptiMEM and cultured in a low-volume of OptiMEM for an additional 24 h, at which point conditioned media was harvested and EV's were isolated.

Isolation and labeling of extracellular vesicles
Media was prepared as described above in 25 mL of media in a T125 cell culture flask.In some cases, bulk extracellular vesicles were isolated by concentrating media in a 100 kDa spin-filter.In other cases, exosomes were specifically isolated from conditioned media using Takara Capturem EV spin columns (Takara, 635723) according to the manufacturer's instructions.For extracellular vesicle labeling experiments, BV-2 microglia were cultured in T25 flasks in 5 mL of Dulbecco's modified eagle medium (DMEM; Gibco) and activated overnight with LPS (1 µg/mL).Supernatant was isolated and incubated with PKH67 dye for 15 minutes at 37 °C following the PKH67GL-1KT kit (Sigma) 19 .Extracellular vesicles were then isolated as described above and resuspended in Neurobasal media (Gibco) with 1% GlutaMAX and 2% B-27 supplement.Primary hippocampal neurons were treated overnight with dyed EV preparation and imaged following kit instructions.GW4869 treatment of microglia GW4869 (#D1692, Sigma) was dissolved in DMSO to make a stock solution of 0.2 mg/mL.For inhibition of exosome generation, BV-2 microglia were treated for 3 µM GW4869 for 24 hours before 24-hour LPS treatment.Culture supernatants were collected for exosome isolation and neuronal treatment.Periodate labeling of cell surface sialosides Cells were washed with HBSS solution (Gibco) and then incubated with 1 mM sodium (meta)periodate (NaIO 4 ; Sigma) in DPBS (Sigma) for 30 minutes on ice.Cells were then washed twice with sodium acetate buffer (pH 4.7) and fixed for 10 minutes in a 1:1 acetate buffer:methanol solution on ice, then fixed for 10 min in pure methanol.Cells were then washed with sodium acetate buffer and incubated with AlexaFluor 488 hydroxylamine dye (25 uM in sodium acetate buffer; Thermo) for 1 hour at 4 Celsius.Imaging was performed on an Axio Observer Z-1 (Zeiss) equipped with a Spectra-X Light engine LED light (Lumencor), controlled with Micro-Manager.Images were acquired with a Plan-Apochromat 20x/0.8air objective (Zeiss) paired with image capture from the ORCA-Flash4.0sCMOS camera (sCMOS; Hamamatsu).Dye was excited with cyan LED 470/24 nm) and emission was collected after passing through the Zeiss 90 HE filter set (425/30 nm, 514/30 nm, 592/25 nm, 709/100 nm LP).

Generation of CRISPR KO of mNeu3
Plasmid constructs encoding Cas9 and a sgRNA were prepared in the lentiCRISPRv2 vector according to published protocols. 1Guides were selected based on the genome-wide guides described by Bassik and coworkers. 2Plasmid-bearing Stbl3 E. coli were grown in 50 mL cultures and DNA was extracted and endotoxin purified by MiraPrep. 3tiCRISPRv2 plasmids were packaged in lentiviruses produced from HEK293Ts cotransfected with pGag/Pol, pRev, pTat, and pVSVG (gift from the Yi-Chang Liu and Jonathan Weismann).In brief, 1.5 µg of LentiCRISPRv2 plasmid were combined with 0.1 µg of each packaging plasmid and Lipofectamine LTX (Thermo Fisher, 15338100) in OptiMEM (Thermo Fisher, 31985062).Transfection complexes were added to HEK293Ts at 70-80% confluency in a 6 well plate in 2 mL fresh media.The media was aspirated after 12 h and bleached.After 48 h, the media was harvested and filtered through a 0.45 µm syringe filter to afford the viral supernatants.BV2 cells were resuspended in fresh viral-containing media with polybrene (8 µg/mL).Media was changed after 24 h, and after 72 h antibiotic selection was started (2.5 µg/mL).After two weeks of selection, editing of the target gene was validated by TIDE analysis. 4okine release assay Adherent NEU3 KO and WT BV-2 microglia were plated (100,00 cells/well in a 24-well plate) in Neurobasal media (Gibco) with 1% GlutaMAX and 2% B-27 supplement one day prior to experiment.Media was treated with LPS (1 μg/mL), LPS + DANA (2 μM), or left untreated during plating.Three technical replicates were made per treatment.After 24 hours, cells were spun down at 500 rcf for 5 minutes to remove debris, and supernatant was extracted for analysis.Cytokine levels were assessed using the BD Cytometric Bead Array (CBA) Mouse Inflammation Kit.Flow cytometry was performed on a BD Accuri C6 Plus, and FlowJo software was used to gate on single cells and live cells for analysis.μL Generation of endogenous tagging of mNeu3 by homology-directed recombination Endogenous tagging of murine Neu3 in BV2 cells was achieved using the Mendenhall-Myers system. 5In brief, a pFETCh donor plasmid (Addgene, 63934) containing homology arms for mNeu3 (see table of gene fragments) was prepared along with PX458 plasmids containing one the only potential target cut site for mNeu3, as outlined by the target selection described by Mendenhall and Myers.Plasmids were prepared from 50 mL cultures of Stellar E. coli and purified by MiraPrep. 3 passage BV2 microglia were transfected by magnetofection (OZ Biosciences) according to the manufacturer's protocols.After 48 h, microglia were treated with a low dose of antibiotic selection (G418, 200X).After two weeks of treatment, only the cells co-transfected with pFETCh donor and PX458 bearing the sgRNA were alive and growing well in the presence of G418 (0.25 mg/mL).The polyclonal population was grown out and the genomic DNA was isolated using a GeneJET Genome DNA Purification Kit (Thermo).PCR was performed using primers +/-750 bp of the insertion site and compared to PCR products from wt cells.A clear 2.5 kbp band was observed in addition to a 1.5 kbp band of comparable intensity, indicating (mostly) monoallelic insertion of the transfer gene.

Dissociated hippocampal cultures
All animal procedures were approved by Stanford University's Administrative Panel on Laboratory Animal Care and conformed to the NIH Guide for Care and Use of Laboratory Animals and the Public Health Policy.Primary hippocampal tissue was harvested from E16.5 C57BL/6 embryonic mice (Charles River) immediately after sacrifice of the pregnant dam.The isolated hippocampi were dissociated using Papain Dissociation System (Worthington Biochemical Corporation) and trituration with fire-polished Pasteur pipettes.The dissociated cells were plated onto 12mm coverslips (Chemglass Life Sciences) pre-treated with Poly-D Lysine (PDL; 1 mg/mL, Sigma-Aldrich) at a density of 6 × 104 cells per coverslip.The cells were maintained for 24 hours in Dulbecco's modified eagle medium (DMEM; Gibco) supplemented with 4.5 g/L D-glucose, 10% FBS, 1% GlutaMAX, and 2% B-27 (Gibco), and then switched to Neurobasal media (Gibco) with 1% GlutaMAX and 2% B-27 supplement.Plates were incubated at 5% CO2, 37°C, and subsequent media changes took place every 7 days.

Voltage imaging of neurons
Neurons were incubated with an HBSS solution (Gibco) containing BeRST1 (1.0µM; Miller Lab, UC Berkeley) for 20 minutes at 37°C prior to imaging.Functional imaging of hippocampal cells was performed on an Axio Observer Z-1 (Zeiss) equipped with a Spectra-X Light engine LED light (Lumencor), controlled with Micro-Manager.Images were acquired with a Plan-Apochromat 20x/0.8air objective (Zeiss) paired with image capture from the ORCA-Flash4.0sCMOS camera (sCMOS; Hamamatsu) with 4x4 in-camera binning.We used a sampling rate of 0.5 kHz over 5 or 10 seconds in order to resolve individual action potentials.To achieve maximum sampling rate, a field of view (FOV) size of 512x100 pixels (665.6x130µm) was used for simultaneous recording of 5-15 neurons at a time.BeRST 1 was excited with a 631 nm light (LED; 631nm, 28 nm bandpass) with an LED power of 70%.Emission from BeRST 1 was collected with a 680/10 nm bandpass emission filter after passing through a dichroic mirror (425/30 nm, 514/30 nm, 592/25 nm, 709/100 nm LP).

Image analysis
Analysis of voltage traces in primary neurons was performed using ImageJ and custom Python scripts.All Python scripts used to analyze data in this study are available at github.com/rishi-kulkarni/SpykeMapper.Regions of interest (ROIs) were drawn around cell bodies within a field of view and the average fluorescence over time was extracted and inputted into an Excel workbook.The fluorescence time course data was then analyzed using a custom Python script that performed subthreshold trace extraction and spike train generation.Briefly, the subthreshold activity was identified using asymmetric least squares regression and subtracted from raw time course data to generate a flattened trace containing a flat baseline and spiking activity.Spikes were identified from the flattened trace using a threshold of +6 STDEV of all cellular fluorescence values within a coverslip to generate a digitized spike train containing all-or-nothing firing data or Raster plot.Factor analysis was performed using the FactoryAnalyzer module with no rotation.The shared variance values per network were compared using Cohen's d.

Preparation of extracellular vesicles for mass spectrometry
Extracellular vesicles were enriched as described above in the section Isolation and labeling of extracellular vesicles.Enriched EVs were labeled with membrane-impermeable sulfo-NHS-biotin (Thermo Fisher Scientific, 21217) per the manufacturer's protocol.In brief, EVs in pH 8.0 PBS were labeled for 30 min at room temperature with sulfo-NHS-biotin (2 mM) and were quenched by the addition of 100 mM glycine.EVs solutions were desalted using spin columns.EVs were lysed by the subsequent addition of 10X RIPA buffer.Samples were prepared in triplicate, and triplicate control samples were treated equivalently, except sulfo-NHS-biotin was excluded.All samples were enriched using streptavidin magnetic beads (Pierce, #88817) and the DynaMag-2 Magnet (Invitrogen, #12321D) based on a protocol adopted from previous work. 6Buffers were made fresh, including ETDA buffer (150 mM NaCl, 100 mM Tris pH 8, 0.5 mM EDTA pH 8), urea buffer (2 < urea, 10 mM Tris) and elution buffer (5% SDS in RIPA, 20 mM DTT, 2 mM biotin). 25 μL of streptavidin beads were used per sample, and beads were washed with 50 μL of cold RIPA twice before being resuspended in 25 μL cold RIPA.Beads were added to each sample, which were continuously rotated overnight at 4 C.The next day, the unbound fraction was removed from the beads and kept.Beads were washed with 100 μL RIPA, which was added to the saved unbound fraction (labeled FT, flow through) for each sample.Next, beads were washed twice with 200 μL RIPA, three times with EDTA buffer, three times with 1.5 M NaCl, three times with 0.1 M NaHCO3, and once with urea buffer.Proteins were eluted from the beads by boiling at 95 C with 60 μL elution buffer for 10 min.The elution step was repeated, for a total of two elution steps, and elutes from each step were combined for a given sample.Digestion was performed for eluted and FT proteins using a micro Strap protocol provided by the manufacturer (Protifi). 7For FT samples, proteins were brought to 5% SDS and reduced with 10 mM DTT for 10 minutes at 95 C. Cysteines were alkylated using 40 mM iodoacetamide for 45 minutes each at room temperature in the dark.The lysate was then acidified with phosphoric acid, brought to approximately 80-90% methanol with 100 mM TEAB in 90% methanol, and loaded onto the S-trap column.Following washing with 100 mM TEAB in 90% methanol, trypsin (Promega) was added to the S-trap at a 20:1 protein:protease ratio for 90 minutes at 47 °C.Peptides were eluted in three steps that were collected in the same tube for a given sample: 40 μL of 50 mM TEAB, 40 μL 0.2% FA in water, and 40 μL of 0.2% FA in 50% ACN, all spun at 4,000 x g for 1 minute.Eluted peptides were dried via lyophilization.Preparation of cell-surface labeled neurons for mass spectrometry Samples were prepared following published cell surface capture protocols that label cell surface proteins through sialic acids. 8,9and lysate protein concentrations were quantitated by BCA (Pierce).Lysates were digested using a mini S-trap protocol (Protifi), which is similar to the micro S-trap protocol above, but with different volumes.Lysates were brought to 5% SDS and reduced with 5 mM DTT for 5 minutes at 95 C. Cysteines were alkylated using 25 mM iodoacetamide for 45 minutes each at room temperature in the dark.Lysates were then acidified with phosphoric acid, brought to approximately 80-90% methanol with 100 mM TEAB in 90% methanol, and loaded onto the S-trap column.Following washing with 100 mM TEAB in 90% methanol, trypsin (Promega) was added to the S-trap at a 20:1 protein:protease ratio for 90 minutes at 47 °C.Peptides were eluted in three steps that were collected in the same tube for a given sample: 80 μL of 50 mM TEAB, 80 μL 0.2% FA in water, and 80 μL of 0.2% FA in 50% ACN, all spun at 4,000 x g for 1 minute.Eluted peptides were dried via lyophilization and dried peptides were then resuspended in 100 μL 100 mM Tris, pH 8 for enrichment via streptavidin beads (Pierce, #88817) and the DynaMag-2 Magnet (Invitrogen, #12321D).Buffers were made fresh, including ETDA buffer (150 mM NaCl, 100 mM Tris pH 8, 0.5 mM EDTA pH 8).50 μL of streptavidin beads were used per sample, and beads were washed with 200 μL 100 mM Tris three times before being resuspended in 400 μL 100 mM Tris.Beads were added to each sample (500 μL total per sample), which were continuously rotated overnight at 4 C.The next day, the unbound fraction was removed from the beads and kept.Beads were washed with 500 μL 100 mM Tris, which was added to the saved unbound fraction (labeled FT, flow through) for each sample.Next, beads were washed five times with 500 μL 100 mM Tris, five times with 500 μL EDTA buffer, five times with500 μL 1.5 M NaCl, five times with 500 μL 0.1 M NaHCO3, once with 500 μL 80% (v/v) 2-isopropanol, twice with500 μL water, and three times with 500 μL warm (60 °C) 100 mM Tris.Beads were then resuspended in 500 μL 100 mM Tris.Glycerol-free PNGaseF (New England Biolabs, # P0705L) was diluted 2-fold, and 1 μL of diluted PNGaseF was added to each set of beads.Beads with PNGaseF were incubated overnight, where PNGaseF enzymatic cleavgage release formerly N-glycosylated peptides (i.e., de-glycopeptides).Eluted de-glycopeptides were acidified with 10% FA before desalting with Strata-X reversed phase SPE cartridges (Phenomenex, #8B-S100-AAK) by conditioning the cartridge with 1 mL ACN followed by 1 mL 0.2% formic acid (FA) in water.Acidified de-glycopeptides loaded on to the cartridge, followed by a 1 mL wash with 0.2% FA in water.Peptides were eluted with 400 μL of 0.2% FA in 80% ACN and dried via lyophilization.Mass spectrometry proteomics LC-MS/MS had more than three missing values.Missing values for the remaining identifications were imputed from a normal distribution with width 0.3 and downshift value of 1.8 (i.e., default values).

Statistical analysis
All statistical hypothesis tests were performed using either a hierarchical permutation test 16

Figure S1 .
Figure S1.Tracking of Indels by Decomposition (TIDE) analysis of NEU3 KO BV-2 microglia.To confirm knock-out of the NEU3 gene in murine BV-2 microglia, CRISPR-Cas9 edited cells were subjected to two weeks of selection before genomic DNA was harvested and the cut region was amplified and sequenced.TIDE [ref] deconvolutes the possible changes from CRISPR-based editing to provide gene-level editing quantiation.

Figure S7 .
Figure S7.Neu3 on HeLa-derived extracellular vesicles is sufficient to desialylate neurons in culture.Primary mouse hippocampal neurons were treated with conditioned media from NEU3-overexpressing HeLa cells in the presence or absence of deoxy-2,3-anhydroneuraminic acid (DANA).Cell surface sialic acid levels were visualized by periodate labeling.Representative images (A) and quantification of fluorescence (B) reveal that Neu3 from HeLa-derived extracellular vesicles significantly reduce surface sialic acids (p=0.028) in a sialidase-inhibitor dependent manner.n=3 coverslips/condition, 25 cells/condition.
Figure S8.Neu3 on HeLa-derived extracellular vesicles do not produce significant change in firing rate of neurons.Neurons were labeled with the voltage-sensitive dye BeRST1 and treated with extracellularvesicle enriched media from either microglia or Neu3 over-expressing cells.Neuronal firing rates and network connectivity were analyzed by fluorescence microscopy.(A,B) BeRST1 is a membrane-localizing voltagesensitive fluorophore that undergoes a dramatic increase in fluorescence intensity in response to changes in membrane potential, i.e. upon the depolarization of firing neurons.Representative brightfield and BeRST1 fluorescence of a single field of view (A) and voltage traces of each neuron in a single field of view (B) contain both subthreshold activity and spiking activity.(C) BV-2 microglia treated with or without LPS and with or without GW4869.The EVs from the conditioned media were enriched and neurons were treated with EVenriched media, and neuronal activity was measured by voltage imaging with BeRST1.Firing rates of neurons treated with BV-2 EV-enriched media reveal 1.7 Hz decrease in +LPS condition compared to -LPS condition (p=0.048).The effect is partially rescued by inhibition of EV production with GW4869 (+LPS vs. +LPS+GW4869, p=0.062).(D) As in (C), but using conditioned media from HeLa cells overexpressing either wild-type or loss-of-function (Y369F) Neu3.Firing rates of neurons treated with EV-enriched media of NEU3overexpressing HeLa cells reveal no significant changes between functional NEU3 and a loss-of-function point mutant (WT: -0.26 Hz, p=0.58;Y369F: -0.14 Hz, p=0.65).For (C): n=4 coverslips/condition, 168 neurons total.For (D): n=3 coverslips/condition, 331 total neurons.All hypothesis testing was performed by hierarchical permutation tests.